RESUMEN
Multipotent stromal cells (MSC) demonstrate remarkable functional heterogeneity; however, its molecular mechanisms remain largely obscure. In this study, we explored MSC response to hormones, which activate Gs-protein / cyclic AMP (cAMP) / protein kinase A (PKA) dependent signaling, at the single cell level using genetically encoded biosensor PKA-Spark. For the first time, we demonstrated that about half of cultured MSCs are not able to activate the cAMP/PKA pathway, possibly due to the limited availability of adenylyl cyclases. Using this approach, we showed that MSC subpopulations responding to various hormones largely overlapped, and the share of responding cells did not exceed 40%. Using clonal analysis, we showed that signaling heterogeneity of MSC could be formed de novo within 2 weeks.
Asunto(s)
Adenilil Ciclasas/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/clasificación , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Hormonas/farmacología , Células Madre Mesenquimatosas/metabolismo , Adenilil Ciclasas/genética , Células Cultivadas , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Humanos , Células Madre Mesenquimatosas/efectos de los fármacos , Transducción de SeñalRESUMEN
Primary adipose tissue-derived multipotent stem/stromal cells (adMSCs) demonstrate unusual signaling regulatory mechanisms, i.e., increased of sensitivity to catecholamines in response to noradrenaline. This phenomenon is called "heterologous sensitization", and was previously found only in embryonic cells. Since further elucidation of the molecular mechanisms that are responsible for such sensitization in primary adMSCs was difficult due to the high heterogeneity in adrenergic receptor expression, we employed immortalized adipose-derived mesenchymal stem cell lines (hTERT-MSCs). Using flow cytometry and immunofluorescence microscopy, we demonstrated that the proportion of cells expressing adrenergic receptor isoforms does not differ significantly in hTERT-MSCs cells compared to the primary adMSCs culture. However, using analysis of Ca2+-mobilization in single cells, we found that these cells did not demonstrate the sensitization seen in primary adMSCs. Consistently, these cells did not activate cAMP synthesis in response to noradrenaline. These data indicate that immortalized adipose-derived mesenchymal stem cell lines demonstrated impaired ability to respond to noradrenaline compared to primary adMSCs. These data draw attention to the usage of immortalized cells for MSCs-based regenerative medicine, especially in the field of pharmacology.
Asunto(s)
Agonistas alfa-Adrenérgicos/farmacología , Células Madre Mesenquimatosas/efectos de los fármacos , Norepinefrina/farmacología , Tejido Adiposo/citología , Señalización del Calcio , Línea Celular , Células Cultivadas , AMP Cíclico/metabolismo , Humanos , Células Madre Mesenquimatosas/metabolismoRESUMEN
A series of hitherto unknown hetaryl-substituted (in phosphonium part) phosphonium-iodonium ylides were synthesized. The reaction of these mixed phosphonium-iodonium ylides with acetylenes opens a way to new furyl annelated phosphinolines or unusually substituted phosphininofurans.